Hereditary spherocytosis, elliptocytosis, stomatocytosis, acanthocytosis, xerocyto-sis, and pyropoikilocytosis can be diagnosed on the basis of their characteristic morphologic abnormalities. Spectrin is responsible for maintaining red cell shape and is composed of two subunits, a- and P-spectrin, which are structurally distinct and are encoded by separate genes. A variety of mutations in a- and P-spectrin have been reported. Spectrin regulates the lateral mobility of integral membrane proteins and provides structural support for the lipid bilayer. Disruption of spectrin self-association leads to disorders characterized by abnormally shaped red cells. Enzyme defects and many hemoglobinopathies have nonspecific morphologic abnormalities.
Table 7-4. Tests Used to Demonstrate a Hemolytic Process
Accelerated hemoglobin catabolism Serum bilirubin level Urinary urobilinogen excretion Fecal urobilinogen excretion Haptoglobin level Plasma hemoglobin level Methemoglobin level Methemalbumin level Carboxyhemoglobin
Reticulocyte count/reticulocyte index
Bone marrow examination for erythroid hyperplasia Radiography: hair-on-end appearance
Table 7-5. Tests Used to Establish a Specific Cause of Hemolytic Anemia
Blood smear: spherocytes, ovalocytes, pyknocytes, stomatocytes" Osmotic fragility (fresh and incubated)" Autohemolysis" Cation permeability studies Membrane phospholipid composition Scanning electron microscopy Hemoglobin defects
Blood smear: sickle cells, target cells (Hb C)" Sickling test"
Quantitative fetal hemoglobin determination"
Heat stability test for unstable hemoglobin
Oxygen dissociation curves
Rates of synthesis of polypeptide chain production Fingerprinting of hemoglobin Enzyme defects
Heinz-body preparation" Osmotic fragility" Autohemolysis test" Screening test for enzyme deficiencies" Specific enzyme assays" Extracorpuscular defects
Coombs' test: IgG (gamma), C'3 (complement), broad-spectrum (both gamma and complement)" Acidified serum lysis (Ham's) test" Donath-Landsteiner test"
Flow cytometric analysis of red cells with monoclonal antibodies to GPl-linked surface antigens (for PNH)
"Tests commonly employed and most useful in establishing a diagnosis.
1. Autosomal dominant inheritance (75% of cases). The severity of anemia and the degree of spherocytosis may not be uniform within an affected family.
2. No family history in 25% of cases. Some show minor laboratory abnormalities, suggesting a carrier (recessive) state. Others are due to a de novo mutation.
3. Most common in people of northern European heritage, with an incidence of 1 in 5000.
In hereditary spherocytosis (HS), the primary defect is membrane instability due to dysfunction or deficiency of a red cell skeletal protein. A variety of membrane skeletal protein defects have been found in different families. These include:
1. Ankyrin mutations: Account for 50-67% of HS. In many patients, both spectrin and ankyrin proteins are deficient. Mutations of ankyrin occur in both dominant and recessive forms of HS. Clinically, the course varies from mild to severe. Red cells are typically spherocytes.
2. a-Spectrin mutations occur in recessive HS and account for less than 5% of HS. Clinical course is severe. Contracted cells, poikilocytes, and spherocytes are seen.
3. P-Spectrin mutations occur in dominant HS and account for 15-20% of HS. Clinical course is mild to moderate. Acanthocytes, spherocytic elliptocytes, and spherocytes are seen.
4. Protein 4.2 mutations occur in the recessive form of HS and account for less than 5% of HS. Clinical course is mild to moderate. Spherocytes, acanthocytes, and ovalocytes are seen.
5. Band 3 mutations occur in the dominant form of HS and account for 15-20% of HS. Clinical course can be mild to moderate. Spherocytes are occasionally mushroom-shaped or pincered cells.
Deficiency of these membrane skeletal proteins in HS results in vertical defect, which causes progressive loss of membrane lipid and surface area. The loss of surface area results in characteristic microspherocytic morphology of HS red cells.
The sequelae are as follows:
1. Sequestration of red cells in the spleen (due to reduced erythrocyte deforma-bility)
2. Depletion of membrane lipid
3. Decrease in membrane surface area relative to volume, resulting in a decrease in surface area-to-volume ratio
4. Tendency to spherocytosis
5. Influx and efflux of sodium increased; cell dehydration
6. Rapid adenosine triphosphate (ATP) utilization and increased glycolysis
7. Premature red cell destruction.
1. Anemia: Mild to moderate in compensated cases. In erythroblastopenic crisis, hemoglobin may drop to 2-3 g/dL.
2. MCV usually decreased; mean corpuscular hemoglobin concentration (MCHC) raised and RDW elevated.*
3. Reticulocytosis (3-15%).
4. Blood film: Microspherocytes+ (vary in number); hyperdense cells,* polychro-masia.
5. Coombs' test negative.
6. Increased red cell osmotic fragility (spherocytes lyse in higher concentrations of saline than normal red cells) occasionally only demonstrated after incubation of blood sample at 37°C for 24 hours. In spite of normal osmotic fragility, increased MCHC or an increase of hyperdense red cells is highly suggestive of HS.
7. Autohemolysis at 24 and 48 hours increased, corrected by the addition of glucose.
8. Survival of 51Cr-labeled cells reduced with increased splenic sequestration.
9. Marrow: Normoblastic hyperplasia; increased iron.
*The MCHC is only raised in hereditary spherocytosis, hereditary xerocytosis, hereditary pyropoikilo-cytosis, and cold agglutinin disease. The presence of elevated RDW and MCHC (performed by aperture impedance instruments, e.g., Coulter) makes the likelihood of hereditary spherocytosis very high, because these two tests used together are very specific for hereditary spherocytosis.
+The percentage of microspherocytes is the best indicator of the severity of the disease but not a good discriminator of the HS genotype.
iHyperdense cells are seen in HbSC disease, HbCC disease, and xerocytosis. In HS, hyperdense cells are a poor indicator of disease severity but an effective discriminating feature of the HS phenotype.
1. Raised bilirubin, mainly indirect reacting
2. Obstructive jaundice with increased direct-reacting bilirubin; may develop due to gallstones, a consequence of increased pigment excretion.
1. Anemia and jaundice: Severity depends on rate of hemolysis, degree of compensation of anemia by reticulocytosis, and ability of liver to conjugate and excrete indirect hyperbilirubinemia.
3. Presents in newborn (50% of cases) with hyperbilirubinemia, reticulocytosis, normoblastosis, spherocytosis, negative Coombs' test, and splenomegaly.
4. Presents before puberty in most patients.
5. Diagnosis sometimes made much later in life by chance.
6. Co-inheritance of HS with hemoglobin S-C disease may increase the risk of splenic sequestration crisis.
7. Co-inheritance of P-thalassemia trait and HS may worsen, improve, or have no effect on the clinical course of HS.
8. Iron deficiency may correct the laboratory values but not the red cell life span in HS patients.
9. HS with other system involvement:
a. Interstitial deletion of chromosome 8p11.1-8p21.1 causes ankyrin deficiency, psychomotor retardation, and hypogonadism.
b. HS may be associated with neurologic abnormalities such as cerebellar disturbances, muscle atrophy, and a tabes-like syndrome.
Table 7-6 lists a classification of hereditary spherocytosis in accordance with clinical severity and indications for splenectomy.
1. Clinical features and family history
2. Hematologic features.
1. Hemolytic crisis: With more pronounced jaundice due to accelerated hemolysis (may be precipitated by infection)
2. Erythroblastopenic crisis: Dramatic fall in hemoglobin level (and reticulocyte count); usually due to maturation arrest and often associated with giant pronormoblasts in the recovery phase; usually associated with parvovirus B19 infection*
3. Folate deficiency: Caused by increased red cell turnover; may lead to superimposed megaloblastic anemia. Megaloblastic anemia may mask HS morphology as well as its diagnosis by osmotic fragility
*Parvovirus B19 infects developing normoblasts, causing a transient cessation of production. The virus specifically infects CFU-E and prevents their maturation. Giant pronormoblasts are seen in bone marrow. Diagnosis is made by increased IgM antibody titer against parvovirus and PCR for parvovirus on bone marrow.
Table 7-6. Classification of Spherocytosis and Indications for Splenectomy c>
Table 7-6. Classification of Spherocytosis and Indications for Splenectomy
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